Preliminary study on purification and identification of aromatic acid amino L-DOPA from Malaysia freshwater green mussel byssus

Abstract

L-DOPA (L-3, 4-dihydroxyphenylalanine) is a type of aromatic amino acid which can be detected by using acidic extraction and purification method involving adhesive byssus green mussel protein. The main objective of this study is to identify and purify the aromatic amino acid L-DOPA via the utilization of gel Sephadex G-200 filtration chromatrography based on two types of acidic and basic mobile phase solution. The crushing and homogenizing for adhesive byssus green mussel were conducted using a mortar and a pestle with the aid of liquid nitrogen. The samples that had been crushed were then mixed and dissolved in perchloric acid 0.7%, 1.0% and 1.5% (v/v) (pre-treatment) prior to the extraction process. The extraction was carried out by centrifuging the extracts at 11,000 rpm for about 10 mins and at a temperature of 10 °C to obtain supernatant S1. The supernatant was mixed with acetone and sulphuric acid and centrifuged for the second time to produce a pellet and then it was dissolved in the respective mobile phase solutions prior to purification process. Purification was later performed using two mobile phase solutions which were acetic acid 5% (v/v) and NaOH 1M. The absorbance (abs) value of each purified protein extract fractions was collected and analysed at 214 nm to 400 nm with the help of UV-spectrophotometer. The highest abs value was selected for identification and verification of amino acid L-DOPA in the purified solution. Verification was carried out by utilizing high performance liquid chromatography (HPLC) and thin layer chromatrography (TLC). The results showed that the use of 0.7% (v/v) perchloric acid and 5% (v/v) acetic acid for pre-treatment process and mobile phase solution of purification process respectively, yielded the highest effluent abs profile at a wavelength of 260 nm. TLC analysis proved the existence of several important amino acids besides L-DOPA which were tyrosine and phenylalanine after 78 hrs of collection of effluents. Meanwhile, the analysis of HPLC revealed the highest concentration of amino acid L-DOPA (p<0.05) as compared to the other collected effluents.